CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional Application Ser. No. 60/808,834 filed May 26, 2006.
FIELD OF THE INVENTION
- Top of Page
The present invention relates to transgenic corn event MON89034 and plant parts and seed thereof. The event exhibits resistance to insect infestation from insects in the order Lepidoptera. The invention also relates to methods for using plants and seeds comprising DNA that is diagnostic for the presence of the transgenic event when probed for the presence of nucleotide sequences that are unique to the transgenic event, and to methods for detecting the presence of said corn event in a biological sample by detecting specific nucleotide sequences that are unique to the transgenic event. The invention provides nucleotide sequences that are unique to the event.
- Top of Page
OF THE INVENTION
This invention relates to the Lepidopteran resistant transgenic variety of corn (Zea mays) plant referred to herein as event MON89034, and to unique DNA sequences present that, when detected in any sample or variety of corn, are diagnostic for the presence of the transgenic corn plant event MON89034 in that sample or variety, and also relates to the detection of the transgene/genomic insertion region in corn MON89034, and progeny plants and seeds derived therefrom.
The corn plant event MON89034 is particularly resistant to insects in the Lepidoptera family such as Fall armyworm (Spodoptera frugiperda), European corn borer (Ostrinia nubilalis), corn earworm (Helicoverpa zea), southwestern corn borer (Diatraea grandiosella), and black cutworm (Agrotis ipsilon) and the like, all of which are agronomically important insect pests.
Corn is an important crop and is a primary food source in many areas of the world. Biotechnology methods have been applied to corn for the purpose of improving agronomic traits and the quality of the product. One such agronomic trait is insect resistance, for example, genetically engineered resistance to lepidopteran and coleopteran species that arises in corn plants genetically engineered to contain one or more genes encoding insecticidal agents (see for example, U.S. Pat. No. 6,489,542 and U.S. Pat. No. 6,620,988). It is advantageous to detect the presence of a particular transgenic event in a biological sample in order to determine whether one or more progeny of a sexual cross contains the transgenic material. For example, the detection of the event in a sample is important for licensing purposes, for establishing and maintaining purity standards, important for complying with regulatory agencies, for complying with food ingredient standards, for use in legal proceedings in establishing that one or more particular individuals or entities has been using the particular event without a license from the owner or licensee of any patents directed to the transgenic event, and for insuring compliance with various government regulations and/or laws.
In addition, methods that enable the detection of a particular plant would be helpful when complying with regulations requiring the pre-market approval and labeling of foods derived from the recombinant crop plants. Individuals or entities that are resistant to the presence of a transgenic event in a sample also desire reliable methods for detecting the presence of the transgene in a sample in order for them to be able to capitalize on their business, which takes advantage of an absence of transgenes in their products.
Despite these advantages, it is possible that insects may evolve resistance to plants expressing only one B. thuringiensis δ-endotoxin. Such resistance, should it become widespread, would clearly limit the commercial value of germplasm containing single Bt genes.
One possible way of increasing the effectiveness of insecticidal agents provided via transgenic plants and directed at controlling target insect pests and contemporaneously reducing the likelihood of emergence of insect pests resistant to such insecticidal agents would be to ensure that transgenic crops express high levels of these insecticidal agents, such as Bacillus thuringiensis delta-endotoxins (McGaughey and Whalon (1992), Science 258:1451-55; Roush (1994) Biocontrol. Sci. Technol. 4:501-516). In addition, having a repository of insecticidal genes that are effective against groups of insect pests and which manifest their effects through different modes of action can safeguard against development of resistance. The onset of resistance could be substantially delayed as a result of providing a crop that expresses two or more insecticidal activities exhibiting overlapping toxicity to the same insect species. One means for achieving such dual modes of action could be to provide a plant expressing a Bt gene toxic to a particular insect species along with a dsRNA that is provided for the purpose of targeting for suppression an essential gene of the same insect species targeted by the Bt toxin, the dsRNA eliciting an RNAi response upon ingestion by the target pest, providing a means for redundancy in the event that the insect develops resistance either to the dsRNA or to the Bt gene. Alternatively, co-expression in a plant of two or more insecticidal toxins both toxic to the same insect species but each exhibiting a different mode of effectuating its killing activity, particularly when both are expressed at high levels, provides a means for effective resistance management. Examples of such insecticides useful in such combinations include but are not limited to Bt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins, deallergenized and de-glycosylated patatin proteins and/or permuteins, plant lectins, and the like.
The expression of foreign genes in plants is known to be influenced by their chromosomal position, perhaps due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulation elements (e.g., enhancers) close to the integration site (Weising et al. (19880 Ann. Rev. Genet 22:421-477). For this reason, it is often necessary to screen a large number of events in order to identify an event characterized by optimal expression of an introduced gene of interest. Even then, with dozens or even hundreds of different transgenic events in hand, there is no certainty of success in identifying a single transgenic event that provides the optimum levels of expression of the at least two different toxins or insecticidal agents and lacks any undesirable agronomic deficiencies or phytotoxic effects, either as a result of the insertion into some essential or partially essential region of the plant genome, or as a result of toxic effects brought about by the levels of expression of the transgenes. For example, it has been observed in plants and in other organisms that there may be wide variation in the levels of expression of an introduced gene among events. There may also be differences in spatial or temporal patterns of expression, for example, differences in the relative expression of a transgene in various plant tissues, that may not correspond to the patterns expected from transcriptional regulatory elements present in the introduced gene construct. For this reason, it is common to produce several hundreds to several thousands different events and screen the events for a single event that has the desired transgene expression levels and patterns for commercial purposes. An event that has the desired levels or patterns of transgene expression is useful for introgressing the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are suitably adapted to specific local growing conditions.
It is possible to detect the presence of a transgene by any well known nucleic acid detection method such as the polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, or even the coding sequence encoding the protein or dsRNA of interest expressed from the transgene(s), etc. As a result, such methods may not be useful for discriminating between different events, particularly those produced using the same DNA construct, unless the sequence of chromosomal DNA adjacent to the inserted DNA (“flanking DNA”) is known. Depending on the method used for introducing the transgene(s) into a plant genome, abberant or unusual effects can be observed, which often severely complicate the identification of the plant genome sequences flanking the transgenic DNA that was intended to be introduced into the plant. Often, rearrangements of the inserted DNA, rearrangements of the flanking genome DNA, or rearrangements of both the inserted DNA and the flanking genome DNA are prevalent, and complicate the analysis of the insertional event being evaluated. Therefore, it is advantageous to have a means for selecting, for identifying, and for insuring the purity and characteristics of a particular transgenic event in a sample, and the only way to accomplish this is to identify one or more unique sequences associated only with the desired transgenic event, and the presence of such sequences in a biological sample containing DNA of the plant species into which the transgenic DNA was inserted to give rise to the event are thus diagnostic for the event in such sample.
- Top of Page
OF THE INVENTION
The present invention is related to the transgenic corn plant designated MON89034 and progeny that are indistinguishable from corn event MON89034 (to the extent that they also contain at least one allele that corresponds to the inserted transgenic DNA) thereof having seed deposited on Mar. 28, 2006 with American Type Culture Collection (ATCC) with Accession No. PTA-7455. Another aspect of the invention is the progeny plants, or seeds, or regenerable parts of the plants and seeds of the corn event MON89034 that contain a polynucleotide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. The invention also includes plant parts of the corn event MON89034 that include, but are not limited to pollen, ovule, flowers, shoots, roots, stalks, silks, tassels, ears, and leaves, so long as these parts contain at least the polynucleotides as set forth above. Novel genetic compositions contained in the genome of MON89034 and products such from MON89034 such as meal, flour, oil, pulp, and biomass left over in a field of corn plants corresponding to MON89034 event are an aspect of this invention.
The invention provides an insect resistant corn plant that has all of the physiological and morphological characteristics of the corn event MON89034.
According to one aspect of the invention, compositions and methods are provided for detecting the presence of the transgene/genomic insertion region from a novel corn plant designated MON89034. DNA sequences are provided that comprise at least one junction sequence of MON89034 selected from the group consisting of SEQ ID NO:1 (located at positions 2051 to 2070 on SEQ ID NO: 5) and SEQ ID NO:2 (located at positions 11367 to 11388) and complements thereof; wherein a junction sequence spans the junction between heterologous DNA inserted into the genome and the DNA from the corn cell flanking the insertion site and is diagnostic for the event (FIG. 1). A corn event MON89034 and seed comprising these DNA molecules is an aspect of this invention.
DNA sequences that comprise the novel transgene/genomic insertion region, SEQ ID NO:3 and SEQ ID NO:4 (FIG. 2) from corn event MON89034 are aspects of this invention. The corn plant and seed comprising these molecules are also aspects of this invention.
According to another aspect of the invention, two DNA molecules are provided for use in a DNA detection method, wherein the first DNA molecule comprises at least 11 or more contiguous polynucleotides of any portion of the transgene region of the DNA molecule of SEQ ID NO:3 and a DNA molecule of similar length of any portion of a 5′ flanking corn genomic DNA region of SEQ ID NO:3, where these DNA molecules when used together are useful as DNA primers in a DNA amplification method that produces an amplicon. The amplicon produced using these DNA primers in the DNA amplification method is diagnostic for corn event MON 89304 when the amplicon contains SEQ ID NO:1. Any amplicon produced by DNA primers homologous or complementary to any portion of SEQ ID NO:3 and any amplicon that comprises SEQ ID NO:1 is an aspect of the invention.
According to another aspect of the invention, two DNA molecules are provided for use in a DNA detection method, wherein the first DNA molecule comprises at least 11 or more contiguous polynucleotides of any portion of the transgene region of the DNA molecule of SEQ ID NO:4 and a DNA molecule of similar length of any portion of a 3′ flanking corn genomic DNA of SEQ ID NO:4, where these DNA molecules are useful as DNA primers in a DNA amplification method. The amplicon produced using these DNA primers in the DNA amplification method is diagnostic for corn event MON 89304 when the amplicon contains SEQ ID NO:2. Any amplicons produced by DNA primers homologous or complementary to any portion of SEQ ID NO:4 and any amplicon that comprises SEQ ID NO:2 is an aspect of the invention.
According to another aspect of the invention, methods of detecting the presence of DNA corresponding to the corn event MON89034 in a sample are provided. Such methods comprise: (a) contacting the sample comprising DNA with a primer set that, when used in a nucleic acid amplification reaction with genomic DNA from corn event MON89034, produces an amplicon that is diagnostic for corn event MON89034; (b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and (c) detecting the amplicon wherein said amplicon comprises SEQ ID NO:1 or SEQ ID NO:2.
A corn plant, or seed, or product derived from the plant or seed MON89034 wherein the genomic DNA comprising comprises a DNA molecule consisting essentially of SEQ ID NO:5 and complements thereof. A corn plant, or seed, or product derived from the plant or seed MON89034, in which the genomic DNA when isolated from the corn plant, or seed, or product comprises a DNA molecule incorporating nucleotides 2061 to 11377 of SEQ ID NO:5 and complements thereof.
A corn plant, or seed, or product derived from the plant or seed MON89034, in which the genomic DNA when isolated from the corn plant, or seed, or product produces an amplicon in a DNA amplification method, wherein DNA primer molecules SEQ ID NO:6 and SEQ ID NO:7 is used in the DNA amplification method.
According to another aspect of the invention, methods of detecting the presence of a DNA corresponding to the MON89034 event in a sample, such methods comprising: (a) contacting the sample comprising DNA with a probe that hybridizes under stringent hybridization conditions with genomic DNA from corn event MON89034 and does not hybridize under the stringent hybridization conditions with a control corn plant; (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to the corn event MON89034 DNA wherein said probe comprises SEQ ID NO:1 and SEQ ID NO:2.
Another aspect of the invention is a method of determining the zygosity of the progeny of corn event MON89034 comprising: (a) contacting the sample comprising corn DNA with a primer set comprising SQ2842 (SEQ ID NO:6), SQ2843 (SEQ ID NO:7), SQ6523 (SEQ ID NO:10), SQ6524 (SEQ ID NO:11), PB880 (SEQ ID NO:14) and PB2931 (SEQ ID NO:15) that when used in a nucleic-acid amplification reaction with genomic DNA from corn event MON89034, produces a first amplicon that is diagnostic for corn event MON89034 and (b) performing a nucleic acid amplification reaction, thereby producing the first amplicon; and (c) detecting the first amplicon; and (d) contacting the sample comprising corn DNA with said primer set, that when used in a nucleic-acid amplification reaction with genomic DNA from corn plants produces a second amplicon comprising the native corn genomic DNA homologous to the corn genomic region of a transgene insertion identified as corn event MON89034; and (e) performing a nucleic acid amplification reaction, thereby producing the second amplicon and (f) detecting the second amplicon; and (g) comparing the first and second amplicons in a sample, wherein the presence of both amplicons indicates the sample is heterozygous for the transgene insertion.
One aspect of the invention is providing in the diet of a lepidopteran pest an insecticidally effective amount of corn event MON89034.
Another aspect of the present invention is providing a composition or biological sample in the form of a commodity or foodstuff that is derived from corn event MON89034, the commodity or foodstuff comprising ears of corn, shucked corn, corn silk, corn pollen, cracked corn, corn meal, crushed corn, corn flour, corn oil, corn starch, corn steep liquor, corn malt, corn sugar, corn syrup, margarine produced from corn oil, unsaturated corn oil, saturated corn oil, corn flakes, pop corn, ethanol and/or liquor produced from corn or corn products comprising DNA diagnostic for corn event MON89034, distillers dry goods solids (DDGS) produced from fermentation of such corn event, and animal feeds comprising such DDGS and/or corn, whether or not whole, cracked, or crushed, processed foodstuffs, a cosmetic, and a bulking agent in which there is found a detectable amount of a polynucleotide that is diagnostic for the presence of the transgenic corn event MON89034 in the biological sample. An alternative means for providing corn as a foodstuff is to provide corn in various forms of grain for feeding, such as whole corn, cracked corn, crushed corn, and various forms of the foregoing in a blend with milo, suet, millet, sunflower, oats, wheat, rice, beans, and the like. Detectable amounts of a nucleotide sequence in such commodity or foodstuff, such as is set forth at SEQ ID NO:1 or SEQ ID NO:2, or the complements thereof, is diagnostic for the presence of such transgenic event MON89034 DNA in the sample, and therefore, the presence of the transgenic event cells as having originated the DNA in the sample.
The foregoing and other aspects of the invention will become more apparent from the following detailed description.
FIG. 1. Organization of the transgene insert present within the genome of transgenic corn event MON89034. The central open or white bar represents the inserted DNA. Below the white bar is a diagram that represents the various elements within the inserted DNA. The ends of the inserted DNA have been arbitrarily designated as 5′ (to the left side of the Figure) and 3′ (to the right side of the Figure). The Right Border and Left Border sequences or segments are labeled beneath each end of the diagram illustrating the various elements within the inserted DNA. The labeled elements in the expression cassettes within the inserted DNA are, in consecutive order starting from the Right Border: e35S promoter, wheat CAB untranslated leader, rice actin intron, coding sequence for Cry1A.105, wheat HSP17 3′ termination and polyadenylation sequence, FMV promoter, hsp70 intron, rubisco small subunit chloroplast targeting peptide coding sequence, Cry2Ab coding sequence, nos 3′ termination and polyadenylation signal sequence, and then the Left Border. The vertically hatched bars at either end of the central open or white bar correspond to the arbitrarily labeled 5′ and 3′ corn genome flanking sequences. The longest black line above the hatched and open or white bar represents SEQ ID NO:5 (the full length sequence represented by the figure depicting the 5′ flanking sequence, the inserted DNA sequence, and the 3′ flanking sequence). The shorter black lines above and below the black line labeled as SEQ ID NO:5 represent the approximate positions within SEQ ID NO:5 in which each of the specifically labeled sequences can be found (i.e., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4). SEQ ID NO:1 and SEQ ID NO:2, and any sequence derived from corn event MON89034 containing SEQ ID NO:1 and/or SEQ ID NO:2, are diagnostic for corn event MON89034 DNA in a biological sample.